This U19 Cooperative Agreement grant application is designed to perform preclinical studies to optimize a combination microbicide composed of the HIV-1 entry inhibitor retrocyclin RC101 and the nonnucleoside reverse transcriptase inhibitor (NNRTI) 5-chloro-3-phenylsulfonylindole-2-carboxamide (CSIC). Our goal is to develop a microbicide that will have potent anti-HIV activity against cell-free and cell-associated virus while inducing minimal viral resistance, be non-toxic and non-inflammatory to cervical and vaginal tissues and be safe and effective against vaginal SHIV challenge in monkeys. An optimal combination of RC-101 and CSIC with these properties will be chosen based on a highly interactive approach between four projects and two cores. Together, we will employ an array of in vitro and ex vivo laboratory tests of efficacy and toxicity, in vivo safety and efficacy tests in monkey models, and studies to optimize a silicone elastomer ring formulation of our compounds. The 4 projects of this grant application are: 1) Development of CSIC as a microbicide (Project 1, Dr. Parniak), 2) RC-101 as an intravaginal anti-HIV-1 topical microbicide (Project 2, Dr. Cole), 3) Testing candidate microbicides for antiviral activity and toxicity in a cervical tissue based organ culture (Project 3, Dr. Gupta), and 4) Evaluation of toxicity and efficacy of CSIC and RC-101 in monkeys (Project 4, Dr. Marx). The scientific studies in these four projects will be supported by the Administrative Core (Core A, Dr. Gupta) and a Formulation Core (Core B, Drs. Rohan and Smith). Dr. Tom Smith (Auritec Pharmaceuticals), serving as the Industry partner in this U19 program, will bring controlled release pellet technologies to develop vaginal ring delivery of RC-101- and CSIC-based microbicides in monkeys. In designing the scope of the proposed topical microbicide program, we have focused on extensively evaluating safety and toxicity of candidate microbicides utilizing in vitro studies, tissue explants and animal models, as well as an expanded and innovative organ culture model to study the antiviral activity of microbicides in the presence of common STIs. Our multidisciplinary approach to microbicide development targets two different sites of HIV-1 replication (entry and reverse transcription) through a controlled-release ring formulated product, utilizes a novel ex vivo organ culture to test toxicity, inflammation and antiviral activity across cervicovaginal mucosa, and applies macaque models to study toxicity and efficacy. Therefore, the proposed study will provide novel strategies to develop microbicides for the prevention of HIV-1 transmission and their sequelae for women.